Kaouther Zahra

First report of molecular diagnosis of Tunisian hemophiliacs A: Identification of 8 novel causative mutations

AbstractIntroductionHemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder.AimIn this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum.MethodsWe screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure.ResultsWe identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles.ConclusionThe mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715

Low dose prophylaxis in Tunisian children with haemophilia.

Low dose prophylaxis could be recommended in countries with limited resources.

Identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia

Inherited factor VII (FVII) deficiency is a rare disorder characterized by a bleeding phenotype varying from mild to severe. To date, more than 200 mutations have been described along the F7 gene encoding for FVII. The aim of this study was the identification of genetic defects underlying FVII deficiency in 10 patients belonging to eight unrelated families of the North provinces from Tunisia. Mutation detection was performed by sequencing the whole F7 gene coding region, exon-intron boundaries and about 400 bp of the promoter region. We identified 5 mutations in five unrelated families; the novel p.F328Y mutation and the reported mutations: p.R304Q, p.M298I, IVS1aG > A and p.G-39G. For the remaining 5 patients we didn’t identified any mutations using PCR/Sequencing protocol. In conclusion, this study represents the first comprehensive molecular series of FVII deficiency affected patients in Tunisia from the North. We will try in the future to continue the molecular study for Tunisian patients from Center and South provinces in order to have a complete idea about the FVII deficiency mutational profile in our country.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1288044089753085

Identification of novel and recurrent mutations in Tunisian haemophilia B patients

Haemophilia B disease is a recessively inherited X-linked bleeding disorder which results from deficiency of factor IX (F9). Haemophilia B has a frequency of approximately 1 in 25 000 men worldwide [1]. Haemophilia B results from heterogeneous mutations spread throughout the F9 gene [2]. According to the World Federation of Hemophilia Report on the annual global survey 2007, 51 haemophilia B in Tunisia have been reported [3]. In this first study on Tunisian haemophilia B, we report the molecular analysis of 16 unrelated haemophilia B families. Patients involved in this study were from the Hemophilia Treatment Center, Aziza Othmana hospital, Tunisia. Informed consent was obtained from all patients. Molecular analysis was performed using the following strategy: polymerase chain reactions for the entire coding sequence of the F9 gene were prepared as described previously [4]. The mutation detection protocol was performed by dHPLC on a WAVE DNA Fragment Analysis System (Transgenomics, San Jose, USA). Altered profiles detected by dHPLC were sequenced using ABI Dye Terminator Cycle Sequencing (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) and analysed using a capillary sequencer Genetic Analyser ABI PRISM310 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA [4]. Results were analysed using BLAST (http://www.ncbi.nlm. nih.gov/blast) program against the normal F9 gene sequence (GenBank Accession No. K02402) and the mutations were compared with the haemophilia B mutation database (http://www.umds.ac.uk/molgen). To evaluate the nature of missense mutations, we used PolyPhen (Polymorphism Phenotyping) (http://genetics.bwh.harvard.edu/pph). Our cohort is composed of 30 patients belonging to 16 unrelated families who represent 60% of total haemophilia B Tunisian population. A total of 15 different mutations were detected (Table 1), except for one family that did not show any mutations. In addition, the polymorphism g.20421A>G in exon 6 was also identified in five families. Five novel mutations were identified in five patients including 2 missense mutations, 1 nonsense mutation, 1 splice site mutation and one small deletion. For patient Hb 2, a deletion of CAG sequence from the 17795 to 17797 position inducing the loss of the last acid Ala173 in exon 5 (The numbering of the amino acids is according to the Swiss-Prot PZES (P00740)). For patient Hb10, a T to A substitution at nucleotide position 113 which changes a Cys acid in a codon stop (Cys27X) in exon 1, which will result in nonsense-mediated RNA decay and produce a severe phenotype as no protein will be translated, has been revealed. Patient Hb12 shows an acceptor splice substitution at the position 10507 in intron 4 (+2T > C). For Patient Hb14, a substitution of T to A at the position 31286 in exon 8, change the Cys 435 to Ser. Patient Hb16 present a substitution of G to A at the position 30932 in exon 8, which change Ala 317 to Thr. Replacement of a non-polar amino acid residue by a polar one is likely to affect the function, secretion or stability of the protein. Using PolyPhen these two mutations are predicted to be probably damaging with a score of 1.000 and 0.995 respectively. The question of whether these two candidate mutations Cys435Ser and Ala317Thr are pathogenic and alter the three-dimensional structure and function of F9 protein needs further investigation. However, as the latter mutations along with, Ala173Del, Ala317Thr and Cys435Ser occurred at amino acid residues highly conserved among different species, they may be involved in the F9 destabilization. Compared with previously published reports [5], we found that the two deletions identified in our patients bearing a severe disease. However, in our patient cohort the two nonsense mutations were associated with different phenotypes, severe and moderate disease respectively in patient Hb17(FIX:C level of 4) which is at variance with the majority of entries on the haemophilia B database for this mutation (which cite FIX:C and antigen levels of <1 for most of the 56 examples). Our observation in Hb17 is actually the exception for this particular mutation and for most nonsense mutations in general. In patient Hb3, we could not detect any mutations in the F9 gene using first dHPLC (no altered profile was observed) then sequencing. It is possible that this family might have pathological translocation, duplication or inversion in the factor IX gene leading to the disease. Further investigation is needed. To our knowledge this study is the first comprehensive molecular analysis of haemophilia B patients in Tunisia. Five novel mutations were identified and our data are globally in agreement with other reports in the international database. When requested, the data obtained from this study will be used for carrier testing and prenatal diagnosis. The identification of the mutations can also be used to estimate the risk of inhibitor development. It can also be valuable when planning future studies including gene therapy.

Regional registry of bleeding disorders in Tunisia.

is unlikely that the occurrence of secondary PPH was recorded. Most cases of secondary PPH are dealt within the community or in the emergency setting and therefore a different approach is required to identify its true incidence. Previous case series reported higher risk of secondary PPH in women with VWD [3,6]. The use of pictorial blood loss assessment chart to quantify puerperal loss has demonstrated that women with bleeding disorders have longer duration of lochia (39 days, range 21–58 days) compared with women without a bleeding disorder (31 days, range 10–62 days) P = 0.03 [9]. There is insufficient data on the factor levels in the affected women; among 24 women with type 1 VWD included in the study, at least three had levels above 50 IU dL . It is possible that some of the women included in the study only had borderline levels or very mildly reduced levels and thus not a confirmed diagnosis of VWD. The authors have cited the rise in von Willebrand factor (VWF) and factor VIII (FVIII) levels that occur during pregnancy as an explanation for VWD posing no significant increased risk of primary PPH. In general, patients with type 1 VWD show an increase in their VWF and FVIII levels during pregnancy. Although most women achieve factor levels >50 IU dL 1 by full term, some cases of type 1 VWD have insufficient correction in the haemostatic defect [10], especially those with factor levels that are more than mildly reduced [5]. In type 2A VWD, VWF antigen may increase in pregnancy, but its activity remains unchanged. There may also be worsening thrombocytopaenia in type 2B, and those with type 3 VWD show no increase in their factor levels. Therefore, clinician should be vigilant when managing women with VWD in pregnancy and avoid complacency and reliance on improvements of the coagulation defect. An individualized assessment of the bleeding risk prior to delivery and careful planning for an appropriate haemostatic cover during delivery and postpartum period are essential to reduce the risk of PPH. In summary, it is clear from other studies already published in the literature that the risk of PPH and other bleeding complications are higher in women with VWD. Although the authors of this study state that they found no evidence that women with VWD have a significantly increased risk of PPH, a close look at their findings show higher risk of PPH in VWD and significant increase in the risk for those with confirmed diagnosis before pregnancy.

The Glanzmann’s Thrombasthenia in Tunisia: A Cohort Study

Background The Glanzmann’s thrombasthenia (GT) is a rare autosomal-recessive bleeding disorder with uncommon neonatal revelation. It is due to abnormalities of quantitative and/or qualitative αIIbβ3 integrin. This cell adhesion receptor is essential for platelet aggregation and allows the formation of a hemostatic plug if the vessel is damaged by injury. The clinical picture of GT is variable, with mucocutaneous bleeding due to non-functional platelets. Management requires a good expertise in bleeding disorders. We describe the clinical and the epidemiological data of GT in Aziza Othmana Hospital Hemophilia Center. Methods This was a retrospective study of all patients with GT monitored and treated in our hemophilia center during the period of 2011 - 2015. Results Twenty-seven patients among the 35 patients included in our hemophilia center registry were studied. The most common sign encountered is the gingival bleeding. In our women cohort, one completed her pregnancy. The consanguinity is present with a frequency of 62%. Treatments used depending on the case are tranexamic acid, platelet transfusion, packed red blood cells and rFVIIa, respectively. Conclusion GT is relatively frequent in Tunisia and especially in the North of the country which can be explained by the high consanguinity in our population.